PROJECT SUMMARY/ABSTRACT Scientific Premise and Prior Research: Puncture wounds, be they produced by accident, intent or medical practice, e.g., surgery, can be a routine to major traumatic event. Moreover, drug aggravated bleeding is a major limitation of current antiplatelet drugs that form a cornerstone of cardiovascular patient therapy and a potentially major liability to any surgery. We contend that detailed structure understanding of the platelet centric responses that occur within the actual puncture hole is essential to fill a void in prior research and yield an important framework for the management traumatic injuries and common bleeding side effects. Gap(s) in knowledge: Previous approaches to thrombus formation have focused mostly on examples in which the exposed vascular matrix remains intact and therefore little to no bleeding occurs and have suffered from the inability of 2P microscopy or SEM to assess platelet activation at the level of individual platelets. From this circumscribed experimental perspective, a ?Core and Shell? model of thrombus structure, i.e., a horizontal core of matrix anchored, highly activated platelets covered by a shell of weakly activated platelets in which signaling within the Core is thrombin-dependent and within the Shell is ADP/thromboxane-dependent has emerged. We submit that this model may well be limited in its application to actual bleeding where the wound is an open puncture hole. Solution to problem: Taking alternate visualization approaches that determine the activation state of individual platelets in 3D space, our Preliminary Studies have already yielded data redefining normal, bleeding cessation as exemplified in a puncture wound model. Our data reveal a spatially dispersed, platelet activation pattern and strong indications that initial steps in thrombus formation are dependent on the capture of platelet aggregates that cap the hole from the outside rather than fill the hole. This has led us to formulate a new ?Cap and Build? paradigm. How both platelet-centric and coagulation factor cascade events can be integrated within the paradigm is now an important question. To understand these multiple inputs through experimentation, we have proposed Aims that focus first on signaling events, be they autocrine (platelet secretion, Specific Aim 1) or exocrine (as inferred from the effects of anti-platelet drugs or DOACs, Specific Aim 2), and the importance of platelet adhesion receptors (Specific Aim 3). We also propose to test how well the Cap and Build paradigm apply to smaller, less traumatic injury.

R01HL155519

2021-09-01

2025-05-30